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Stratagene e coli xl1 blue cells
Isotype-specific phage enrichment during subsequent rounds of affinity selection. The number of phage was determined as colony-forming units (CFU) after infection of freshly grown Escherichia coli <t>XL1</t> Blue cells. Notably, the absolute number of eluted phage significantly depends on the isotype library used for screening.
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1) Product Images from "Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin"

Article Title: Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin

Journal: Immunology

doi: 10.1046/j.1365-2567.2003.01728.x

Isotype-specific phage enrichment during subsequent rounds of affinity selection. The number of phage was determined as colony-forming units (CFU) after infection of freshly grown Escherichia coli XL1 Blue cells. Notably, the absolute number of eluted phage significantly depends on the isotype library used for screening.
Figure Legend Snippet: Isotype-specific phage enrichment during subsequent rounds of affinity selection. The number of phage was determined as colony-forming units (CFU) after infection of freshly grown Escherichia coli XL1 Blue cells. Notably, the absolute number of eluted phage significantly depends on the isotype library used for screening.

Techniques Used: Selection, Infection

2) Product Images from "Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin"

Article Title: Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin

Journal: Immunology

doi: 10.1046/j.1365-2567.2003.01728.x

Isotype-specific phage enrichment during subsequent rounds of affinity selection. The number of phage was determined as colony-forming units (CFU) after infection of freshly grown Escherichia coli XL1 Blue cells. Notably, the absolute number of eluted phage significantly depends on the isotype library used for screening.
Figure Legend Snippet: Isotype-specific phage enrichment during subsequent rounds of affinity selection. The number of phage was determined as colony-forming units (CFU) after infection of freshly grown Escherichia coli XL1 Blue cells. Notably, the absolute number of eluted phage significantly depends on the isotype library used for screening.

Techniques Used: Selection, Infection

3) Product Images from "Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors"

Article Title: Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors

Journal: PLoS Biology

doi: 10.1371/journal.pbio.0050007

Phenotype of E. coli XL1-Blue Strain Expressing the Selected Inhibitory DARPins Six AcrB inhibitors (2907_5, 1108_5, 1108_6, 1108_9, 1108_19, and A11), one unselected nonbinding DARPin (E3_5), and an acrB knockout strain (KAM3 [ 14 ], transformed also with E3_5) were ranked for their R6G sensitivity. Induction of the DARPins results in the inhibition of AcrB, yielding an R6G-sensitive phenotype. E. coli cells were transformed with the respective plasmid and plated under different conditions: nonselective, no R6G or no IPTG; selective where different R6G concentrations create an environment in which inhibitory DARPins repress growth. All plates contained IPTG (except plate no IPTG). The plates were colored with Coomassie brilliant blue for better visibility of the colonies.
Figure Legend Snippet: Phenotype of E. coli XL1-Blue Strain Expressing the Selected Inhibitory DARPins Six AcrB inhibitors (2907_5, 1108_5, 1108_6, 1108_9, 1108_19, and A11), one unselected nonbinding DARPin (E3_5), and an acrB knockout strain (KAM3 [ 14 ], transformed also with E3_5) were ranked for their R6G sensitivity. Induction of the DARPins results in the inhibition of AcrB, yielding an R6G-sensitive phenotype. E. coli cells were transformed with the respective plasmid and plated under different conditions: nonselective, no R6G or no IPTG; selective where different R6G concentrations create an environment in which inhibitory DARPins repress growth. All plates contained IPTG (except plate no IPTG). The plates were colored with Coomassie brilliant blue for better visibility of the colonies.

Techniques Used: Expressing, Knock-Out, Transformation Assay, Inhibition, Plasmid Preparation

4) Product Images from "Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease"

Article Title: Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2006.02.049

Insertion of human FLJ20160 protein into membranes of E. coli or P. pastoris . Membrane fractions were separated from cytoplasm by centrifugation and samples containing 200 μg (a) or 50 μg (b) of protein were subjected to SDS/polyacrylamide gel (12%) electrophoresis. The gel was electroblotted to PVDF membrane and probed with HRP-conjugated antibody directed against His-tag. Molecular weight ladder is shown in kDa. (a) Membrane (lane 1) or cytoplasm (lane 2) fractions isolated from control untransformed E. coli XL1-Blue cells. Membrane (lane 3) or cytoplasm (lane 4) fractions isolated from E. coli XL1-Blue cells harboring pSP72 plasmid encoding human FLJ20160 protein. Purified LacY/His10 (0.2 μg; lane 5) is shown for comparison. (b) Membrane fractions isolated from different P. pastoris strains harboring pPICZ-A plasmid encoding human FLJ20160 protein after induction with methanol. KM71H (lane 1), X-33 (lane 2), and GS115 (lane 3).
Figure Legend Snippet: Insertion of human FLJ20160 protein into membranes of E. coli or P. pastoris . Membrane fractions were separated from cytoplasm by centrifugation and samples containing 200 μg (a) or 50 μg (b) of protein were subjected to SDS/polyacrylamide gel (12%) electrophoresis. The gel was electroblotted to PVDF membrane and probed with HRP-conjugated antibody directed against His-tag. Molecular weight ladder is shown in kDa. (a) Membrane (lane 1) or cytoplasm (lane 2) fractions isolated from control untransformed E. coli XL1-Blue cells. Membrane (lane 3) or cytoplasm (lane 4) fractions isolated from E. coli XL1-Blue cells harboring pSP72 plasmid encoding human FLJ20160 protein. Purified LacY/His10 (0.2 μg; lane 5) is shown for comparison. (b) Membrane fractions isolated from different P. pastoris strains harboring pPICZ-A plasmid encoding human FLJ20160 protein after induction with methanol. KM71H (lane 1), X-33 (lane 2), and GS115 (lane 3).

Techniques Used: Centrifugation, Electrophoresis, Molecular Weight, Isolation, Plasmid Preparation, Purification

Related Articles

Clone Assay:

Article Title: Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa †
Article Snippet: Paragraph title: Phage DNA isolation, cloning, and sequencing. ... After E. coli XL1-Blue cells (Stratagene) were transformed and plated on Luria-Bertani agar containing ampicillin (50 μg/ml; Fisher Scientific, Hanover Park, Ill.), X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (80 μg/ml; Fisher Scientific), and IPTG (isopropyl-β- d -thiogalactoside) (20 mM; Fisher Scientific), white colonies were purified, cultures were grown, and plasmid DNA was isolated using an UltraClean plasmid mini-prep kit (Mo Bio Laboratories, Solana Beach, Calif.).

Article Title: Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin
Article Snippet: .. Nine different isotype-specific ligation mixtures were used for transformation of E. coli XL1 Blue cells (Stratagene, LaJolla, CA) yielding libraries containing between 1 × 107 and 5 × 108 primary clones. .. For screening, the libraries harbouring the VH chains of the IgM, IgA and IgG isotype were pooled to obtain libraries only distinguished by the isotype of the heavy chain, but not by the light chain (these libraries were formally termed A, G and M, and isolated single clones were labelled with the letter of the library followed by a number, e.g. A2 for clone IgA no. 2).

Article Title: A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies
Article Snippet: .. The mutant clones and wild-type Z13e1 Fab were transformed separately into Escherichia coli XL1-Blue cells (Stratagene), and single colonies were used to inoculate 10 ml super broth medium containing 50 μg/ml of carbenicillin and 10 μg/ml of tetracycline. .. Cell pellets were lysed by being resuspended in 1 ml CelLytic B buffer (Sigma) and incubated for 20 min at room temperature.

Article Title: Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors
Article Snippet: For this purpose, the selected pool of binders was cloned into the vector pQIA [ ]. .. The pool of binders was transformed into E. coli XL1-blue cells (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]; Stratagene, http://www.stratagene.com ) and plated under nonselective conditions (ampicillin, IPTG, no R6G).

Article Title: Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease
Article Snippet: Paragraph title: Cloning end expression of human FLJ20160 gene ... E. coli XL1-Blue cells (Stratagene, La Jolla, CA) transformed with pSP72 plasmid encoding the FLJ20160 gene were grown in LB medium at 37 °C with 100 mg/l ampicillin.

Article Title: Crystal structure of Trbp111: a structure-specific tRNA-binding protein
Article Snippet: The reaction mixture was then purified on a QiaQuick (Qiagen, Santa Clarita, CA) column and introduced into E.coli XL1-blue cells (Stratagene) by heat shock transformation. .. The PCR-amplified coding sequence for the E.coli homolog was cloned into the PET21b vector (Novagen, Madison, WI) so as to fuse a sequence encoding a His6 tag to the C-terminal end of the Trbp111 coding sequence.

Centrifugation:

Article Title: Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa †
Article Snippet: After the agar overlay was harvested and subjected to centrifugation to remove agar and cell debris, the supernatant was treated with DNase and RNase (Sigma, St. Louis, Mo.) and the phage particles were concentrated by precipitation with polyethylene glycol 8000 (Fisher Biotech) and resuspended in SM buffer ( ). .. After E. coli XL1-Blue cells (Stratagene) were transformed and plated on Luria-Bertani agar containing ampicillin (50 μg/ml; Fisher Scientific, Hanover Park, Ill.), X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (80 μg/ml; Fisher Scientific), and IPTG (isopropyl-β- d -thiogalactoside) (20 mM; Fisher Scientific), white colonies were purified, cultures were grown, and plasmid DNA was isolated using an UltraClean plasmid mini-prep kit (Mo Bio Laboratories, Solana Beach, Calif.).

Article Title: Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease
Article Snippet: E. coli XL1-Blue cells (Stratagene, La Jolla, CA) transformed with pSP72 plasmid encoding the FLJ20160 gene were grown in LB medium at 37 °C with 100 mg/l ampicillin. .. Cells were induced with 0.3 mM IPTG, harvested after 4 h by centrifugation and disrupted by using an Emulsiflex (Avestin International, Canada).

Amplification:

Article Title: Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin
Article Snippet: Polymerase chain reaction (PCR) amplification of the immunoglobulin VH and VL genes was carried out in a total reaction volume of 50 µl, with 1 ng of cDNA as template and a set of specific primers, as described in ref. . .. Nine different isotype-specific ligation mixtures were used for transformation of E. coli XL1 Blue cells (Stratagene, LaJolla, CA) yielding libraries containing between 1 × 107 and 5 × 108 primary clones.

Article Title: Engineering synthetic antibody inhibitors specific for LD2 or LD4 motifs of paxillin
Article Snippet: .. Pulled down PMPs with bound phages were used to infect E. coli XL1-Blue cells (Stratagene) for overnight phage amplification at 37°C. .. The amplified phages were submitted to additional three rounds of biopanning, in which phage solution in TBST-BSA containing MBP was incubated separately with free MBP-LD2 or MBP-LD4.

Article Title: Crystal structure of Trbp111: a structure-specific tRNA-binding protein
Article Snippet: Following 18 cycles of PCR amplification, the reaction was incubated with Dpn 1 to nick the plasmid DNA. .. The reaction mixture was then purified on a QiaQuick (Qiagen, Santa Clarita, CA) column and introduced into E.coli XL1-blue cells (Stratagene) by heat shock transformation.

Mass Spectrometry:

Article Title: Characterization of Human Type C Enterotoxin Produced by Clinical S. epidermidis Isolates
Article Snippet: Dendograms were generated from main spectra projection using MALDI/TOF MS Microflex™ system and Biotyper™ software. .. Escherichia coli XL1 blue cells [recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac (F′ proAB lacIqZΔM15 Tn10 (tet r))] (Stratagene, Agilent technologies, Massie, France) were used as recipient cells for transformation with recombinant pGEX-6P-1- sec epi gene (GE healthcare life Science, Buc, France).

Positive Control:

Article Title: Characterization of Human Type C Enterotoxin Produced by Clinical S. epidermidis Isolates
Article Snippet: S. aureus SCP FRI 361 strain, a SEC producer was used as a positive control to verify the purified recombinant protein identity. .. Escherichia coli XL1 blue cells [recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac (F′ proAB lacIqZΔM15 Tn10 (tet r))] (Stratagene, Agilent technologies, Massie, France) were used as recipient cells for transformation with recombinant pGEX-6P-1- sec epi gene (GE healthcare life Science, Buc, France).

Construct:

Article Title: Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes
Article Snippet: 2.6 Construction and Characterization of the E35Q/H300N Double Mutant The E35Q/H300N double mutant was constructed by combining the pGTacStEH1-5H-based mutants E35Q and H300N. .. The plasmid was transformed into Escherichia coli XL1-Blue cells (Stratagene).

Article Title: A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies
Article Snippet: Z13e1 Fab mutants were constructed in phagemid cloning vector pComb3X, which encodes the wild-type Z13e1 light and heavy chains, by use of the QuikChange XL mutagenesis kit (Stratagene). .. The mutant clones and wild-type Z13e1 Fab were transformed separately into Escherichia coli XL1-Blue cells (Stratagene), and single colonies were used to inoculate 10 ml super broth medium containing 50 μg/ml of carbenicillin and 10 μg/ml of tetracycline.

Article Title: Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease
Article Snippet: The final construct contained the full-length human gene encoding FLJ20160 under the LacZ promoter/operator and used for expression experiments. .. E. coli XL1-Blue cells (Stratagene, La Jolla, CA) transformed with pSP72 plasmid encoding the FLJ20160 gene were grown in LB medium at 37 °C with 100 mg/l ampicillin.

Article Title: Crystal structure of Trbp111: a structure-specific tRNA-binding protein
Article Snippet: Point mutants in the gene for Trbp111 were constructed by PCR mutagenesis using two complementary DNA oligonucleotides containing the desired mutation according to the QuickChange site-directed mutagenesis method (Stratagene, La Jolla, CA). .. The reaction mixture was then purified on a QiaQuick (Qiagen, Santa Clarita, CA) column and introduced into E.coli XL1-blue cells (Stratagene) by heat shock transformation.

Enzyme-linked Immunosorbent Assay:

Article Title: A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies
Article Snippet: The mutant clones and wild-type Z13e1 Fab were transformed separately into Escherichia coli XL1-Blue cells (Stratagene), and single colonies were used to inoculate 10 ml super broth medium containing 50 μg/ml of carbenicillin and 10 μg/ml of tetracycline. .. Triplicate crude Fab supernatants were prepared to lessen the effect of batch variation and used directly for enzyme-linked immunosorbent assay (ELISA), as described below.

Incubation:

Article Title: A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies
Article Snippet: The mutant clones and wild-type Z13e1 Fab were transformed separately into Escherichia coli XL1-Blue cells (Stratagene), and single colonies were used to inoculate 10 ml super broth medium containing 50 μg/ml of carbenicillin and 10 μg/ml of tetracycline. .. Cell pellets were lysed by being resuspended in 1 ml CelLytic B buffer (Sigma) and incubated for 20 min at room temperature.

Article Title: GroEL binds a late folding intermediate of phage P22 coat protein
Article Snippet: Escherichia coli XL1-Blue cells from Stratagene (La Jolla, CA, USA) containing the plasmid pSigE that carries the GroEL/S operon in pSE380 (Stratagene) were grown at 37°C to a concentration of 4 × 108 cells/mL. .. After incubation overnight, the cultures were spun briefly in a GSA rotor at 10 000 rpm to pellet the cells.

Article Title: Engineering synthetic antibody inhibitors specific for LD2 or LD4 motifs of paxillin
Article Snippet: After 1 h incubation with mixing, the supernatant containing unbound phages was discarded and PMPs were gently washed twice with fresh portion of TBST-BSA containing free MBP. .. Pulled down PMPs with bound phages were used to infect E. coli XL1-Blue cells (Stratagene) for overnight phage amplification at 37°C.

Article Title: Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair
Article Snippet: Diluted samples were plated on minimal media lacking the appropriate amino acids, and colonies were counted after incubation at 30°C for 4-5 days. .. To test the accuracy of the repair DNA from single yeast transformants was isolated as described , and this was used to transform E. coli XL1-Blue cells (Stratagene) to ampicillin resistance.

Article Title: Crystal structure of Trbp111: a structure-specific tRNA-binding protein
Article Snippet: Following 18 cycles of PCR amplification, the reaction was incubated with Dpn 1 to nick the plasmid DNA. .. The reaction mixture was then purified on a QiaQuick (Qiagen, Santa Clarita, CA) column and introduced into E.coli XL1-blue cells (Stratagene) by heat shock transformation.

Cell Culture:

Article Title: Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin
Article Snippet: .. Bound phage were eluted with 0·5 ml of 0·1- m HCl (pH 2·2 adjusted with solid glycine), neutralized with 60 µl of 1- m Tris–HCl (pH 8·0) and used for infection of freshly cultured, exponentially growing E. coli XL1 Blue cells. .. The titre of the eluted phage was determined in CFU by spreading serial dilutions of the freshly infected bacteria onto agar plates containing ampicillin and tetracyline.

Expressing:

Article Title: Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors
Article Snippet: This vector carries an expression cassette for the selected DARPins under the control of an IPTG-inducible T5 promoter. .. The pool of binders was transformed into E. coli XL1-blue cells (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]; Stratagene, http://www.stratagene.com ) and plated under nonselective conditions (ampicillin, IPTG, no R6G).

Article Title: Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease
Article Snippet: Paragraph title: Cloning end expression of human FLJ20160 gene ... E. coli XL1-Blue cells (Stratagene, La Jolla, CA) transformed with pSP72 plasmid encoding the FLJ20160 gene were grown in LB medium at 37 °C with 100 mg/l ampicillin.

BIA-KA:

Article Title: Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease
Article Snippet: E. coli XL1-Blue cells (Stratagene, La Jolla, CA) transformed with pSP72 plasmid encoding the FLJ20160 gene were grown in LB medium at 37 °C with 100 mg/l ampicillin. .. Protein concentrations were measured by using the Micro BCA method (Pierce, Rockford, IL).

Western Blot:

Article Title: Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease
Article Snippet: E. coli XL1-Blue cells (Stratagene, La Jolla, CA) transformed with pSP72 plasmid encoding the FLJ20160 gene were grown in LB medium at 37 °C with 100 mg/l ampicillin. .. Membranes were separated from cytoplasm by centrifugation and subjected to SDS/polyacrylamide gel electrophoresis and Western blot analysis using His-tag directed HRP-conjugated antibody (Qiagen, Valencia, CA), as described.

Article Title: Characterization of Human Type C Enterotoxin Produced by Clinical S. epidermidis Isolates
Article Snippet: Each S. epidermidis isolate was previously screened for SEA, SEB, SEC, SEG, and SEH by Western blot and immunodiffusion techniques [ ]. .. Escherichia coli XL1 blue cells [recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac (F′ proAB lacIqZΔM15 Tn10 (tet r))] (Stratagene, Agilent technologies, Massie, France) were used as recipient cells for transformation with recombinant pGEX-6P-1- sec epi gene (GE healthcare life Science, Buc, France).

Transformation Assay:

Article Title: Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa †
Article Snippet: .. After E. coli XL1-Blue cells (Stratagene) were transformed and plated on Luria-Bertani agar containing ampicillin (50 μg/ml; Fisher Scientific, Hanover Park, Ill.), X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (80 μg/ml; Fisher Scientific), and IPTG (isopropyl-β- d -thiogalactoside) (20 mM; Fisher Scientific), white colonies were purified, cultures were grown, and plasmid DNA was isolated using an UltraClean plasmid mini-prep kit (Mo Bio Laboratories, Solana Beach, Calif.). .. Plasmid inserts were sequenced from both ends using standard T3 and T7 primers and Dye Terminator chemistry (PE Applied Biosystems, Foster City, Calif.) on ABI Prism 373 and 377 automated DNA sequencers (PE Applied Biosystems).

Article Title: Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes
Article Snippet: .. The plasmid was transformed into Escherichia coli XL1-Blue cells (Stratagene). .. The recombinant protein was produced and then purified by Ni(II)-IMAC and size-exclusion chromatography as previously described.

Article Title: Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin
Article Snippet: .. Nine different isotype-specific ligation mixtures were used for transformation of E. coli XL1 Blue cells (Stratagene, LaJolla, CA) yielding libraries containing between 1 × 107 and 5 × 108 primary clones. .. For screening, the libraries harbouring the VH chains of the IgM, IgA and IgG isotype were pooled to obtain libraries only distinguished by the isotype of the heavy chain, but not by the light chain (these libraries were formally termed A, G and M, and isolated single clones were labelled with the letter of the library followed by a number, e.g. A2 for clone IgA no. 2).

Article Title: A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies
Article Snippet: .. The mutant clones and wild-type Z13e1 Fab were transformed separately into Escherichia coli XL1-Blue cells (Stratagene), and single colonies were used to inoculate 10 ml super broth medium containing 50 μg/ml of carbenicillin and 10 μg/ml of tetracycline. .. Cell pellets were lysed by being resuspended in 1 ml CelLytic B buffer (Sigma) and incubated for 20 min at room temperature.

Article Title: Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors
Article Snippet: .. The pool of binders was transformed into E. coli XL1-blue cells (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]; Stratagene, http://www.stratagene.com ) and plated under nonselective conditions (ampicillin, IPTG, no R6G). ..

Article Title: Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease
Article Snippet: .. E. coli XL1-Blue cells (Stratagene, La Jolla, CA) transformed with pSP72 plasmid encoding the FLJ20160 gene were grown in LB medium at 37 °C with 100 mg/l ampicillin. .. Cells were induced with 0.3 mM IPTG, harvested after 4 h by centrifugation and disrupted by using an Emulsiflex (Avestin International, Canada).

Article Title: Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair
Article Snippet: In parallel, the same amount of cells was transformed with the same amount of uncut plasmid to normalize for differences in transformation efficiency. .. To test the accuracy of the repair DNA from single yeast transformants was isolated as described , and this was used to transform E. coli XL1-Blue cells (Stratagene) to ampicillin resistance.

Article Title: Crystal structure of Trbp111: a structure-specific tRNA-binding protein
Article Snippet: .. The reaction mixture was then purified on a QiaQuick (Qiagen, Santa Clarita, CA) column and introduced into E.coli XL1-blue cells (Stratagene) by heat shock transformation. ..

Article Title: Characterization of Human Type C Enterotoxin Produced by Clinical S. epidermidis Isolates
Article Snippet: .. Escherichia coli XL1 blue cells [recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac (F′ proAB lacIqZΔM15 Tn10 (tet r))] (Stratagene, Agilent technologies, Massie, France) were used as recipient cells for transformation with recombinant pGEX-6P-1- sec epi gene (GE healthcare life Science, Buc, France). .. Escherichia coli BL21 [F-, ompT , hsdS (rB -, mB -), gal ] was used for over-expression of the glutathione-S-transferase (GST)-S. epidermidis enterotoxin C fusion gene, according to the manufacturer’s (GE healthcare).

Over Expression:

Article Title: Retrieving Biological Activity from LukF-PV Mutants Combined with Different S Components Implies Compatibility between the Stem Domains of These Staphylococcal Bicomponent Leucotoxins
Article Snippet: Escherichia coli XL1 Blue cells ( recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac [F′ proAB lacI q Z ΔM15 Tn 10 ] [ tet r ]) (Stratagene, Amsterdam, The Netherlands) were used as recipient cells after site-directed mutagenesis of recombinant plasmids. .. E. coli BL21 [F− ompT hsdS ( rB − mB − ) gal ] was used for overexpression of the pGEX-6P-1 glutathione S -transferase (GST)-fusioned leucotoxins as recommended by the manufacturer (Pharmacia, Uppsala, Sweden) ( ).

Article Title: Characterization of Human Type C Enterotoxin Produced by Clinical S. epidermidis Isolates
Article Snippet: Escherichia coli XL1 blue cells [recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac (F′ proAB lacIqZΔM15 Tn10 (tet r))] (Stratagene, Agilent technologies, Massie, France) were used as recipient cells for transformation with recombinant pGEX-6P-1- sec epi gene (GE healthcare life Science, Buc, France). .. Escherichia coli BL21 [F-, ompT , hsdS (rB -, mB -), gal ] was used for over-expression of the glutathione-S-transferase (GST)-S. epidermidis enterotoxin C fusion gene, according to the manufacturer’s (GE healthcare).

Crystallization Assay:

Article Title: Crystal structure of Trbp111: a structure-specific tRNA-binding protein
Article Snippet: Wild-type A.aeolicus Trbp111 for crystallization and biochemical assays was purified from TG1 cells (Amersham, Arlington Heights, IL) containing plasmid pTrbp111 as described in . .. The reaction mixture was then purified on a QiaQuick (Qiagen, Santa Clarita, CA) column and introduced into E.coli XL1-blue cells (Stratagene) by heat shock transformation.

Immunodiffusion:

Article Title: Characterization of Human Type C Enterotoxin Produced by Clinical S. epidermidis Isolates
Article Snippet: Each S. epidermidis isolate was previously screened for SEA, SEB, SEC, SEG, and SEH by Western blot and immunodiffusion techniques [ ]. .. Escherichia coli XL1 blue cells [recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac (F′ proAB lacIqZΔM15 Tn10 (tet r))] (Stratagene, Agilent technologies, Massie, France) were used as recipient cells for transformation with recombinant pGEX-6P-1- sec epi gene (GE healthcare life Science, Buc, France).

Ligation:

Article Title: Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin
Article Snippet: .. Nine different isotype-specific ligation mixtures were used for transformation of E. coli XL1 Blue cells (Stratagene, LaJolla, CA) yielding libraries containing between 1 × 107 and 5 × 108 primary clones. .. For screening, the libraries harbouring the VH chains of the IgM, IgA and IgG isotype were pooled to obtain libraries only distinguished by the isotype of the heavy chain, but not by the light chain (these libraries were formally termed A, G and M, and isolated single clones were labelled with the letter of the library followed by a number, e.g. A2 for clone IgA no. 2).

Serial Dilution:

Article Title: Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair
Article Snippet: For galactose-inducible HO sensitivity experiments, cells were grown in the preinduction medium (YP plus 3% glycerol) first, then cultures were diluted to A600 of 0.7, and 5-fold serial dilution were plated either on YPA plates containing glucose or galactose. .. To test the accuracy of the repair DNA from single yeast transformants was isolated as described , and this was used to transform E. coli XL1-Blue cells (Stratagene) to ampicillin resistance.

Infection:

Article Title: Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin
Article Snippet: .. Bound phage were eluted with 0·5 ml of 0·1- m HCl (pH 2·2 adjusted with solid glycine), neutralized with 60 µl of 1- m Tris–HCl (pH 8·0) and used for infection of freshly cultured, exponentially growing E. coli XL1 Blue cells. .. The titre of the eluted phage was determined in CFU by spreading serial dilutions of the freshly infected bacteria onto agar plates containing ampicillin and tetracyline.

SPR Assay:

Article Title: Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors
Article Snippet: The pool of binders was transformed into E. coli XL1-blue cells (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]; Stratagene, http://www.stratagene.com ) and plated under nonselective conditions (ampicillin, IPTG, no R6G). .. The characterization of these DARPins included sequencing, expression tests, surface plasmon resonance, MIC determination, size exclusion chromatography studies, and cocrystallization.

Generated:

Article Title: Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin
Article Snippet: First, VL sublibraries were generated, DNA was isolated from phagemid, restricted with Mlu I/ Not I and used to clone the VH repertoires in a second step. .. Nine different isotype-specific ligation mixtures were used for transformation of E. coli XL1 Blue cells (Stratagene, LaJolla, CA) yielding libraries containing between 1 × 107 and 5 × 108 primary clones.

Article Title: Characterization of Human Type C Enterotoxin Produced by Clinical S. epidermidis Isolates
Article Snippet: Dendograms were generated from main spectra projection using MALDI/TOF MS Microflex™ system and Biotyper™ software. .. Escherichia coli XL1 blue cells [recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac (F′ proAB lacIqZΔM15 Tn10 (tet r))] (Stratagene, Agilent technologies, Massie, France) were used as recipient cells for transformation with recombinant pGEX-6P-1- sec epi gene (GE healthcare life Science, Buc, France).

Polymerase Chain Reaction:

Article Title: Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa †
Article Snippet: The phage DNA was sheared, treated with Bal 31 exonuclease (Promega, Madison, Wis.), and fractionated on a 0.7% SeaPlaque agarose gel (FMC Corporation, Newark, Del.), and fragments in the 1.6- to 3-kb range were purified using a Qiaex II gel extraction kit (QIAGEN, Valencia, Calif.) ( ) and cloned into pPCR-Script Amp using a PCR-Script cloning kit (Stratagene). .. After E. coli XL1-Blue cells (Stratagene) were transformed and plated on Luria-Bertani agar containing ampicillin (50 μg/ml; Fisher Scientific, Hanover Park, Ill.), X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (80 μg/ml; Fisher Scientific), and IPTG (isopropyl-β- d -thiogalactoside) (20 mM; Fisher Scientific), white colonies were purified, cultures were grown, and plasmid DNA was isolated using an UltraClean plasmid mini-prep kit (Mo Bio Laboratories, Solana Beach, Calif.).

Article Title: Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin
Article Snippet: Approximately 20 ng of each first PCR product was used as template for the introduction of flanking restriction sites in a subsequent PCR. .. Nine different isotype-specific ligation mixtures were used for transformation of E. coli XL1 Blue cells (Stratagene, LaJolla, CA) yielding libraries containing between 1 × 107 and 5 × 108 primary clones.

Article Title: Crystal structure of Trbp111: a structure-specific tRNA-binding protein
Article Snippet: Following 18 cycles of PCR amplification, the reaction was incubated with Dpn 1 to nick the plasmid DNA. .. The reaction mixture was then purified on a QiaQuick (Qiagen, Santa Clarita, CA) column and introduced into E.coli XL1-blue cells (Stratagene) by heat shock transformation.

Binding Assay:

Article Title: Engineering synthetic antibody inhibitors specific for LD2 or LD4 motifs of paxillin
Article Snippet: The solution containing unbound fraction was discarded and PMPs with bound proteins were re-suspended in TBST-BSA containing 5 μM biotin to block unoccupied streptavidin and limit non-specific binding. .. Pulled down PMPs with bound phages were used to infect E. coli XL1-Blue cells (Stratagene) for overnight phage amplification at 37°C.

DNA Extraction:

Article Title: Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa †
Article Snippet: Paragraph title: Phage DNA isolation, cloning, and sequencing. ... After E. coli XL1-Blue cells (Stratagene) were transformed and plated on Luria-Bertani agar containing ampicillin (50 μg/ml; Fisher Scientific, Hanover Park, Ill.), X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (80 μg/ml; Fisher Scientific), and IPTG (isopropyl-β- d -thiogalactoside) (20 mM; Fisher Scientific), white colonies were purified, cultures were grown, and plasmid DNA was isolated using an UltraClean plasmid mini-prep kit (Mo Bio Laboratories, Solana Beach, Calif.).

Nucleic Acid Electrophoresis:

Article Title: Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease
Article Snippet: E. coli XL1-Blue cells (Stratagene, La Jolla, CA) transformed with pSP72 plasmid encoding the FLJ20160 gene were grown in LB medium at 37 °C with 100 mg/l ampicillin. .. Membranes were separated from cytoplasm by centrifugation and subjected to SDS/polyacrylamide gel electrophoresis and Western blot analysis using His-tag directed HRP-conjugated antibody (Qiagen, Valencia, CA), as described.

In Vivo:

Article Title: Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors
Article Snippet: Paragraph title: In vivo selection by replica plating. ... The pool of binders was transformed into E. coli XL1-blue cells (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]; Stratagene, http://www.stratagene.com ) and plated under nonselective conditions (ampicillin, IPTG, no R6G).

Mutagenesis:

Article Title: Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes
Article Snippet: Paragraph title: Construction and Characterization of the E35Q/H300N Double Mutant ... The plasmid was transformed into Escherichia coli XL1-Blue cells (Stratagene).

Article Title: A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies
Article Snippet: .. The mutant clones and wild-type Z13e1 Fab were transformed separately into Escherichia coli XL1-Blue cells (Stratagene), and single colonies were used to inoculate 10 ml super broth medium containing 50 μg/ml of carbenicillin and 10 μg/ml of tetracycline. .. Cell pellets were lysed by being resuspended in 1 ml CelLytic B buffer (Sigma) and incubated for 20 min at room temperature.

Article Title: Retrieving Biological Activity from LukF-PV Mutants Combined with Different S Components Implies Compatibility between the Stem Domains of These Staphylococcal Bicomponent Leucotoxins
Article Snippet: .. Escherichia coli XL1 Blue cells ( recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac [F′ proAB lacI q Z ΔM15 Tn 10 ] [ tet r ]) (Stratagene, Amsterdam, The Netherlands) were used as recipient cells after site-directed mutagenesis of recombinant plasmids. .. E. coli BL21 [F− ompT hsdS ( rB − mB − ) gal ] was used for overexpression of the pGEX-6P-1 glutathione S -transferase (GST)-fusioned leucotoxins as recommended by the manufacturer (Pharmacia, Uppsala, Sweden) ( ).

Article Title: Crystal structure of Trbp111: a structure-specific tRNA-binding protein
Article Snippet: Paragraph title: Preparation of wild-type and mutant Trbp111 ... The reaction mixture was then purified on a QiaQuick (Qiagen, Santa Clarita, CA) column and introduced into E.coli XL1-blue cells (Stratagene) by heat shock transformation.

Isolation:

Article Title: Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa †
Article Snippet: .. After E. coli XL1-Blue cells (Stratagene) were transformed and plated on Luria-Bertani agar containing ampicillin (50 μg/ml; Fisher Scientific, Hanover Park, Ill.), X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (80 μg/ml; Fisher Scientific), and IPTG (isopropyl-β- d -thiogalactoside) (20 mM; Fisher Scientific), white colonies were purified, cultures were grown, and plasmid DNA was isolated using an UltraClean plasmid mini-prep kit (Mo Bio Laboratories, Solana Beach, Calif.). .. Plasmid inserts were sequenced from both ends using standard T3 and T7 primers and Dye Terminator chemistry (PE Applied Biosystems, Foster City, Calif.) on ABI Prism 373 and 377 automated DNA sequencers (PE Applied Biosystems).

Article Title: Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin
Article Snippet: Paragraph title: Isolation of peripheral blood mononuclear cells, mRNA and library construction ... Nine different isotype-specific ligation mixtures were used for transformation of E. coli XL1 Blue cells (Stratagene, LaJolla, CA) yielding libraries containing between 1 × 107 and 5 × 108 primary clones.

Article Title: Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease
Article Snippet: The human gene isolated with KpnI/XhoI restriction enzymes from pSP72 plasmid harboring the FLJ20160 gene was ligated into the KpnI/XhoI sites of the yeast expression vector pPICZ-A in-frame with a Myc-epitope and a 6-His-tag. .. E. coli XL1-Blue cells (Stratagene, La Jolla, CA) transformed with pSP72 plasmid encoding the FLJ20160 gene were grown in LB medium at 37 °C with 100 mg/l ampicillin.

Article Title: Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair
Article Snippet: .. To test the accuracy of the repair DNA from single yeast transformants was isolated as described , and this was used to transform E. coli XL1-Blue cells (Stratagene) to ampicillin resistance. .. Plasmid DNA was then isolated and analyzed by restriction enzyme digestion.

Size-exclusion Chromatography:

Article Title: Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes
Article Snippet: The plasmid was transformed into Escherichia coli XL1-Blue cells (Stratagene). .. The recombinant protein was produced and then purified by Ni(II)-IMAC and size-exclusion chromatography as previously described.

Article Title: Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors
Article Snippet: The pool of binders was transformed into E. coli XL1-blue cells (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]; Stratagene, http://www.stratagene.com ) and plated under nonselective conditions (ampicillin, IPTG, no R6G). .. The characterization of these DARPins included sequencing, expression tests, surface plasmon resonance, MIC determination, size exclusion chromatography studies, and cocrystallization.

Article Title: Characterization of Human Type C Enterotoxin Produced by Clinical S. epidermidis Isolates
Article Snippet: .. Escherichia coli XL1 blue cells [recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac (F′ proAB lacIqZΔM15 Tn10 (tet r))] (Stratagene, Agilent technologies, Massie, France) were used as recipient cells for transformation with recombinant pGEX-6P-1- sec epi gene (GE healthcare life Science, Buc, France). .. Escherichia coli BL21 [F-, ompT , hsdS (rB -, mB -), gal ] was used for over-expression of the glutathione-S-transferase (GST)-S. epidermidis enterotoxin C fusion gene, according to the manufacturer’s (GE healthcare).

Purification:

Article Title: Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa †
Article Snippet: .. After E. coli XL1-Blue cells (Stratagene) were transformed and plated on Luria-Bertani agar containing ampicillin (50 μg/ml; Fisher Scientific, Hanover Park, Ill.), X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (80 μg/ml; Fisher Scientific), and IPTG (isopropyl-β- d -thiogalactoside) (20 mM; Fisher Scientific), white colonies were purified, cultures were grown, and plasmid DNA was isolated using an UltraClean plasmid mini-prep kit (Mo Bio Laboratories, Solana Beach, Calif.). .. Plasmid inserts were sequenced from both ends using standard T3 and T7 primers and Dye Terminator chemistry (PE Applied Biosystems, Foster City, Calif.) on ABI Prism 373 and 377 automated DNA sequencers (PE Applied Biosystems).

Article Title: Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes
Article Snippet: The plasmid was transformed into Escherichia coli XL1-Blue cells (Stratagene). .. The recombinant protein was produced and then purified by Ni(II)-IMAC and size-exclusion chromatography as previously described.

Article Title: GroEL binds a late folding intermediate of phage P22 coat protein
Article Snippet: Paragraph title: Purification of GroEL/S ... Escherichia coli XL1-Blue cells from Stratagene (La Jolla, CA, USA) containing the plasmid pSigE that carries the GroEL/S operon in pSE380 (Stratagene) were grown at 37°C to a concentration of 4 × 108 cells/mL.

Article Title: Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase
Article Snippet: .. Human GSTM1 was heterologously expressed from the pKK-D vector in E. coli XL1-Blue cells (Strategene, La Jolla, CA) and purified by affinity chromatography as described previously . .. Human GSTP1 and GSTT1 were expressed and purified as described previously .

Article Title: Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease
Article Snippet: Fragment no. 1 digested with KpnI and BclI and fragment no. 2 digested with BclI and XhoI were purified on agarose gels and sequentially ligated into plasmid pSP72, which confers ampicillin resistance, and contains KpnI, BclI and XhoI restriction sites followed by an in-frame 6-His tag. .. E. coli XL1-Blue cells (Stratagene, La Jolla, CA) transformed with pSP72 plasmid encoding the FLJ20160 gene were grown in LB medium at 37 °C with 100 mg/l ampicillin.

Article Title: Crystal structure of Trbp111: a structure-specific tRNA-binding protein
Article Snippet: .. The reaction mixture was then purified on a QiaQuick (Qiagen, Santa Clarita, CA) column and introduced into E.coli XL1-blue cells (Stratagene) by heat shock transformation. ..

Article Title: Characterization of Human Type C Enterotoxin Produced by Clinical S. epidermidis Isolates
Article Snippet: S. aureus SCP FRI 361 strain, a SEC producer was used as a positive control to verify the purified recombinant protein identity. .. Escherichia coli XL1 blue cells [recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac (F′ proAB lacIqZΔM15 Tn10 (tet r))] (Stratagene, Agilent technologies, Massie, France) were used as recipient cells for transformation with recombinant pGEX-6P-1- sec epi gene (GE healthcare life Science, Buc, France).

Sequencing:

Article Title: Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa †
Article Snippet: Paragraph title: Phage DNA isolation, cloning, and sequencing. ... After E. coli XL1-Blue cells (Stratagene) were transformed and plated on Luria-Bertani agar containing ampicillin (50 μg/ml; Fisher Scientific, Hanover Park, Ill.), X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (80 μg/ml; Fisher Scientific), and IPTG (isopropyl-β- d -thiogalactoside) (20 mM; Fisher Scientific), white colonies were purified, cultures were grown, and plasmid DNA was isolated using an UltraClean plasmid mini-prep kit (Mo Bio Laboratories, Solana Beach, Calif.).

Article Title: Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors
Article Snippet: The pool of binders was transformed into E. coli XL1-blue cells (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]; Stratagene, http://www.stratagene.com ) and plated under nonselective conditions (ampicillin, IPTG, no R6G). .. The characterization of these DARPins included sequencing, expression tests, surface plasmon resonance, MIC determination, size exclusion chromatography studies, and cocrystallization.

Article Title: Characterization of Human Type C Enterotoxin Produced by Clinical S. epidermidis Isolates
Article Snippet: Bacterial Strains, Vectors Media, and Growth Conditions S. epidermidis strains were clinical isolates originated from blood samples and identified with MALDI-TOF Biotyper™ (Bruker Daltonics) and 16S rDNA genes sequencing [ ]. .. Escherichia coli XL1 blue cells [recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac (F′ proAB lacIqZΔM15 Tn10 (tet r))] (Stratagene, Agilent technologies, Massie, France) were used as recipient cells for transformation with recombinant pGEX-6P-1- sec epi gene (GE healthcare life Science, Buc, France).

Affinity Chromatography:

Article Title: Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase
Article Snippet: .. Human GSTM1 was heterologously expressed from the pKK-D vector in E. coli XL1-Blue cells (Strategene, La Jolla, CA) and purified by affinity chromatography as described previously . .. Human GSTP1 and GSTT1 were expressed and purified as described previously .

Blocking Assay:

Article Title: Engineering synthetic antibody inhibitors specific for LD2 or LD4 motifs of paxillin
Article Snippet: The solution containing unbound fraction was discarded and PMPs with bound proteins were re-suspended in TBST-BSA containing 5 μM biotin to block unoccupied streptavidin and limit non-specific binding. .. Pulled down PMPs with bound phages were used to infect E. coli XL1-Blue cells (Stratagene) for overnight phage amplification at 37°C.

Gel Extraction:

Article Title: Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa †
Article Snippet: The phage DNA was sheared, treated with Bal 31 exonuclease (Promega, Madison, Wis.), and fractionated on a 0.7% SeaPlaque agarose gel (FMC Corporation, Newark, Del.), and fragments in the 1.6- to 3-kb range were purified using a Qiaex II gel extraction kit (QIAGEN, Valencia, Calif.) ( ) and cloned into pPCR-Script Amp using a PCR-Script cloning kit (Stratagene). .. After E. coli XL1-Blue cells (Stratagene) were transformed and plated on Luria-Bertani agar containing ampicillin (50 μg/ml; Fisher Scientific, Hanover Park, Ill.), X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (80 μg/ml; Fisher Scientific), and IPTG (isopropyl-β- d -thiogalactoside) (20 mM; Fisher Scientific), white colonies were purified, cultures were grown, and plasmid DNA was isolated using an UltraClean plasmid mini-prep kit (Mo Bio Laboratories, Solana Beach, Calif.).

SDS Page:

Article Title: Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase
Article Snippet: Human GSTM1 was heterologously expressed from the pKK-D vector in E. coli XL1-Blue cells (Strategene, La Jolla, CA) and purified by affinity chromatography as described previously . .. The high purity of the enzymes was confirmed by SDS/PAGE stained with Commassie Brilliant Blue R-250.

Plasmid Preparation:

Article Title: Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa †
Article Snippet: .. After E. coli XL1-Blue cells (Stratagene) were transformed and plated on Luria-Bertani agar containing ampicillin (50 μg/ml; Fisher Scientific, Hanover Park, Ill.), X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (80 μg/ml; Fisher Scientific), and IPTG (isopropyl-β- d -thiogalactoside) (20 mM; Fisher Scientific), white colonies were purified, cultures were grown, and plasmid DNA was isolated using an UltraClean plasmid mini-prep kit (Mo Bio Laboratories, Solana Beach, Calif.). .. Plasmid inserts were sequenced from both ends using standard T3 and T7 primers and Dye Terminator chemistry (PE Applied Biosystems, Foster City, Calif.) on ABI Prism 373 and 377 automated DNA sequencers (PE Applied Biosystems).

Article Title: Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes
Article Snippet: .. The plasmid was transformed into Escherichia coli XL1-Blue cells (Stratagene). .. The recombinant protein was produced and then purified by Ni(II)-IMAC and size-exclusion chromatography as previously described.

Article Title: Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin
Article Snippet: Products were digested with either Xho I/ Hin dIII (for VL ) or Mlu I/ Not I (for VH ) and subcloned into appropriately restricted, phosphatase-treated phagemid vector pSEX81 (F. Breitling; GenBank accession number: ), in two steps. .. Nine different isotype-specific ligation mixtures were used for transformation of E. coli XL1 Blue cells (Stratagene, LaJolla, CA) yielding libraries containing between 1 × 107 and 5 × 108 primary clones.

Article Title: A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies
Article Snippet: Z13e1 Fab mutants were constructed in phagemid cloning vector pComb3X, which encodes the wild-type Z13e1 light and heavy chains, by use of the QuikChange XL mutagenesis kit (Stratagene). .. The mutant clones and wild-type Z13e1 Fab were transformed separately into Escherichia coli XL1-Blue cells (Stratagene), and single colonies were used to inoculate 10 ml super broth medium containing 50 μg/ml of carbenicillin and 10 μg/ml of tetracycline.

Article Title: GroEL binds a late folding intermediate of phage P22 coat protein
Article Snippet: .. Escherichia coli XL1-Blue cells from Stratagene (La Jolla, CA, USA) containing the plasmid pSigE that carries the GroEL/S operon in pSE380 (Stratagene) were grown at 37°C to a concentration of 4 × 108 cells/mL. .. GroEL/S overproduction was induced by the addition of isopropyl- d -thiogalactopyranoside to a final concentration of 0.6 mM.

Article Title: Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors
Article Snippet: This vector carries an expression cassette for the selected DARPins under the control of an IPTG-inducible T5 promoter. .. The pool of binders was transformed into E. coli XL1-blue cells (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]; Stratagene, http://www.stratagene.com ) and plated under nonselective conditions (ampicillin, IPTG, no R6G).

Article Title: Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase
Article Snippet: .. Human GSTM1 was heterologously expressed from the pKK-D vector in E. coli XL1-Blue cells (Strategene, La Jolla, CA) and purified by affinity chromatography as described previously . .. Human GSTP1 and GSTT1 were expressed and purified as described previously .

Article Title: Sequence Alignment and Homology Threading Reveals Prokaryotic and Eukaryotic Proteins Similar to Lactose Permease
Article Snippet: .. E. coli XL1-Blue cells (Stratagene, La Jolla, CA) transformed with pSP72 plasmid encoding the FLJ20160 gene were grown in LB medium at 37 °C with 100 mg/l ampicillin. .. Cells were induced with 0.3 mM IPTG, harvested after 4 h by centrifugation and disrupted by using an Emulsiflex (Avestin International, Canada).

Article Title: Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair
Article Snippet: In parallel, the same amount of cells was transformed with the same amount of uncut plasmid to normalize for differences in transformation efficiency. .. To test the accuracy of the repair DNA from single yeast transformants was isolated as described , and this was used to transform E. coli XL1-Blue cells (Stratagene) to ampicillin resistance.

Article Title: Crystal structure of Trbp111: a structure-specific tRNA-binding protein
Article Snippet: Following 18 cycles of PCR amplification, the reaction was incubated with Dpn 1 to nick the plasmid DNA. .. The reaction mixture was then purified on a QiaQuick (Qiagen, Santa Clarita, CA) column and introduced into E.coli XL1-blue cells (Stratagene) by heat shock transformation.

Software:

Article Title: Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa †
Article Snippet: After E. coli XL1-Blue cells (Stratagene) were transformed and plated on Luria-Bertani agar containing ampicillin (50 μg/ml; Fisher Scientific, Hanover Park, Ill.), X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (80 μg/ml; Fisher Scientific), and IPTG (isopropyl-β- d -thiogalactoside) (20 mM; Fisher Scientific), white colonies were purified, cultures were grown, and plasmid DNA was isolated using an UltraClean plasmid mini-prep kit (Mo Bio Laboratories, Solana Beach, Calif.). .. For direct B3 genome sequencing, custom primers were designed from the assembled clone sequences using PrimerSelect software (DNASTAR, Inc., Madison, Wis.), and the sequence was determined using an ABI Prism 377 automated DNA sequencer (PE Applied Biosystems).

Article Title: Characterization of Human Type C Enterotoxin Produced by Clinical S. epidermidis Isolates
Article Snippet: Dendograms were generated from main spectra projection using MALDI/TOF MS Microflex™ system and Biotyper™ software. .. Escherichia coli XL1 blue cells [recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac (F′ proAB lacIqZΔM15 Tn10 (tet r))] (Stratagene, Agilent technologies, Massie, France) were used as recipient cells for transformation with recombinant pGEX-6P-1- sec epi gene (GE healthcare life Science, Buc, France).

Recombinant:

Article Title: Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes
Article Snippet: The plasmid was transformed into Escherichia coli XL1-Blue cells (Stratagene). .. The recombinant protein was produced and then purified by Ni(II)-IMAC and size-exclusion chromatography as previously described.

Article Title: Retrieving Biological Activity from LukF-PV Mutants Combined with Different S Components Implies Compatibility between the Stem Domains of These Staphylococcal Bicomponent Leucotoxins
Article Snippet: .. Escherichia coli XL1 Blue cells ( recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac [F′ proAB lacI q Z ΔM15 Tn 10 ] [ tet r ]) (Stratagene, Amsterdam, The Netherlands) were used as recipient cells after site-directed mutagenesis of recombinant plasmids. .. E. coli BL21 [F− ompT hsdS ( rB − mB − ) gal ] was used for overexpression of the pGEX-6P-1 glutathione S -transferase (GST)-fusioned leucotoxins as recommended by the manufacturer (Pharmacia, Uppsala, Sweden) ( ).

Article Title: Characterization of Human Type C Enterotoxin Produced by Clinical S. epidermidis Isolates
Article Snippet: .. Escherichia coli XL1 blue cells [recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lac (F′ proAB lacIqZΔM15 Tn10 (tet r))] (Stratagene, Agilent technologies, Massie, France) were used as recipient cells for transformation with recombinant pGEX-6P-1- sec epi gene (GE healthcare life Science, Buc, France). .. Escherichia coli BL21 [F-, ompT , hsdS (rB -, mB -), gal ] was used for over-expression of the glutathione-S-transferase (GST)-S. epidermidis enterotoxin C fusion gene, according to the manufacturer’s (GE healthcare).

Agarose Gel Electrophoresis:

Article Title: Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa †
Article Snippet: The phage DNA was sheared, treated with Bal 31 exonuclease (Promega, Madison, Wis.), and fractionated on a 0.7% SeaPlaque agarose gel (FMC Corporation, Newark, Del.), and fragments in the 1.6- to 3-kb range were purified using a Qiaex II gel extraction kit (QIAGEN, Valencia, Calif.) ( ) and cloned into pPCR-Script Amp using a PCR-Script cloning kit (Stratagene). .. After E. coli XL1-Blue cells (Stratagene) were transformed and plated on Luria-Bertani agar containing ampicillin (50 μg/ml; Fisher Scientific, Hanover Park, Ill.), X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (80 μg/ml; Fisher Scientific), and IPTG (isopropyl-β- d -thiogalactoside) (20 mM; Fisher Scientific), white colonies were purified, cultures were grown, and plasmid DNA was isolated using an UltraClean plasmid mini-prep kit (Mo Bio Laboratories, Solana Beach, Calif.).

Selection:

Article Title: Engineering synthetic antibody inhibitors specific for LD2 or LD4 motifs of paxillin
Article Snippet: Paragraph title: Phage display selection ... Pulled down PMPs with bound phages were used to infect E. coli XL1-Blue cells (Stratagene) for overnight phage amplification at 37°C.

Article Title: Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin Inhibitors
Article Snippet: Paragraph title: In vivo selection by replica plating. ... The pool of binders was transformed into E. coli XL1-blue cells (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)]; Stratagene, http://www.stratagene.com ) and plated under nonselective conditions (ampicillin, IPTG, no R6G).

Article Title: Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair
Article Snippet: Briefly, the yeast- Escherichia coli shuttle plasmid pBTM116, which contain s TRP1 for selection in yeast, was digested with the appropriate restriction enzyme to completion, and the enzyme was inactivated by treatment at 65°C for 20 min. One microgram of linearized DNA was used to transform each strain with the lithium acetate method ( ). .. To test the accuracy of the repair DNA from single yeast transformants was isolated as described , and this was used to transform E. coli XL1-Blue cells (Stratagene) to ampicillin resistance.

Ethanol Precipitation:

Article Title: Complete Genomic Sequence of Bacteriophage B3, a Mu-Like Phage of Pseudomonas aeruginosa †
Article Snippet: Phage DNA was then isolated by phenol extraction and ethanol precipitation ( ). .. After E. coli XL1-Blue cells (Stratagene) were transformed and plated on Luria-Bertani agar containing ampicillin (50 μg/ml; Fisher Scientific, Hanover Park, Ill.), X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside) (80 μg/ml; Fisher Scientific), and IPTG (isopropyl-β- d -thiogalactoside) (20 mM; Fisher Scientific), white colonies were purified, cultures were grown, and plasmid DNA was isolated using an UltraClean plasmid mini-prep kit (Mo Bio Laboratories, Solana Beach, Calif.).

Produced:

Article Title: Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes
Article Snippet: The plasmid was transformed into Escherichia coli XL1-Blue cells (Stratagene). .. The recombinant protein was produced and then purified by Ni(II)-IMAC and size-exclusion chromatography as previously described.

Article Title: Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine
Article Snippet: .. P450cam* and WT P450cam were produced in E. coli XL1-Blue cells by standard procedures. .. The proteins were purified by Ni–NTA affinity chromatography in the presence of D-(+)-camphor, followed by repeated ion exchange chromatography on Q-Sepharose until A391 /A280 was ≥1.4 for enzyme used for kinetic measurements and ≥1.1 for EPR studies.

Concentration Assay:

Article Title: GroEL binds a late folding intermediate of phage P22 coat protein
Article Snippet: .. Escherichia coli XL1-Blue cells from Stratagene (La Jolla, CA, USA) containing the plasmid pSigE that carries the GroEL/S operon in pSE380 (Stratagene) were grown at 37°C to a concentration of 4 × 108 cells/mL. .. GroEL/S overproduction was induced by the addition of isopropyl- d -thiogalactopyranoside to a final concentration of 0.6 mM.

Variant Assay:

Article Title: Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes
Article Snippet: The plasmid carrying the H300N mutation was digested with the restriction enzymes Mun I and Spe I (Thermo Scientific), and the resulting fragment was ligated into the corresponding plasmid encoding the E35Q mutant, digested with the same pair of enzymes, to yield the final variant. .. The plasmid was transformed into Escherichia coli XL1-Blue cells (Stratagene).

Staining:

Article Title: Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase
Article Snippet: Human GSTM1 was heterologously expressed from the pKK-D vector in E. coli XL1-Blue cells (Strategene, La Jolla, CA) and purified by affinity chromatography as described previously . .. The high purity of the enzymes was confirmed by SDS/PAGE stained with Commassie Brilliant Blue R-250.

Positron Emission Tomography:

Article Title: Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase
Article Snippet: Enzyme preparation Human GSTA1 was heterologously expressed from the pET-21a (+) vector in E. coli BL-21 DE3 cells (Novagen, Madison, WI) and purified from bacterial lysate using a HiTrap SP cation-exchange column (Amersham Biosciences) as described previously . .. Human GSTM1 was heterologously expressed from the pKK-D vector in E. coli XL1-Blue cells (Strategene, La Jolla, CA) and purified by affinity chromatography as described previously .

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    Stratagene escherichia coli competent cells
    (a) Schematic illustration of the human IFN-α 2B-containing <t>Escherichia</t> coli –BCG shuttle plasmid. The IFN-α 2B coding sequence is placed downstream of BCG α-antigen signal sequence (SS) and influenza virus haemagglutinin epitope tag sequence (T). Expression of the cytokine is driven by the BCG heat shock protein (hsp)60 promoter (black box). The arrow indicates the direction of transcription. The kanamycin resistance cassette (open box) and a mycobacterial as well as an E. coli origin of replication are indicated. The illustration is not to scale. (b) Expression of IFN-α 2B in Western blot of BCG culture supernatant (S) and pellet lysate (P) from human IFN-α 2B BCG recombinant. Purified recombinant human IFN-α 2B is used as a positive control shown on the right. The blot is probed with a specific mouse anti-human IFN-α MoAb. Standard molecular weights are indicated.
    Escherichia Coli Competent Cells, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    escherichia coli competent cells - by Bioz Stars, 2020-04
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    95
    Stratagene e coli xl1 blue
    Low-temperature growth of E. coli transformants harboring the icdII gene. (Upper panel) E. coli <t>XL1-Blue</t> was transformed with pMSF8 or pMS42. (Lower panel) E. coli DEK2004 was used as the host and transformed with the indicated plasmid. pBluescript was used as a vector. E. coli transformants harboring each plasmid were streaked on MOPS-succinate or MOPS-succinate-yeast extract agar plates and incubated at 10 or 15°C. Photographs were taken 2 weeks after the inoculation.
    E Coli Xl1 Blue, supplied by Stratagene, used in various techniques. Bioz Stars score: 95/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 73 article reviews
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    Image Search Results


    (a) Schematic illustration of the human IFN-α 2B-containing Escherichia coli –BCG shuttle plasmid. The IFN-α 2B coding sequence is placed downstream of BCG α-antigen signal sequence (SS) and influenza virus haemagglutinin epitope tag sequence (T). Expression of the cytokine is driven by the BCG heat shock protein (hsp)60 promoter (black box). The arrow indicates the direction of transcription. The kanamycin resistance cassette (open box) and a mycobacterial as well as an E. coli origin of replication are indicated. The illustration is not to scale. (b) Expression of IFN-α 2B in Western blot of BCG culture supernatant (S) and pellet lysate (P) from human IFN-α 2B BCG recombinant. Purified recombinant human IFN-α 2B is used as a positive control shown on the right. The blot is probed with a specific mouse anti-human IFN-α MoAb. Standard molecular weights are indicated.

    Journal: Clinical and Experimental Immunology

    Article Title: Recombinant bacille Calmette-Gu?rin (BCG) expressing human interferon-alpha 2B demonstrates enhanced immunogenicity

    doi: 10.1046/j.1365-2249.2001.01428.x

    Figure Lengend Snippet: (a) Schematic illustration of the human IFN-α 2B-containing Escherichia coli –BCG shuttle plasmid. The IFN-α 2B coding sequence is placed downstream of BCG α-antigen signal sequence (SS) and influenza virus haemagglutinin epitope tag sequence (T). Expression of the cytokine is driven by the BCG heat shock protein (hsp)60 promoter (black box). The arrow indicates the direction of transcription. The kanamycin resistance cassette (open box) and a mycobacterial as well as an E. coli origin of replication are indicated. The illustration is not to scale. (b) Expression of IFN-α 2B in Western blot of BCG culture supernatant (S) and pellet lysate (P) from human IFN-α 2B BCG recombinant. Purified recombinant human IFN-α 2B is used as a positive control shown on the right. The blot is probed with a specific mouse anti-human IFN-α MoAb. Standard molecular weights are indicated.

    Article Snippet: Escherichia coli competent cells (XL1-Blue MR) obtained from Stratagene (La Jolla, CA) were transformed with the E. coli –BCG shuttle plasmid according to the manufacturer's instructions and selected on kanamycin (30 μg/ml) Luria-Bertani agar plates.

    Techniques: Plasmid Preparation, Sequencing, Expressing, Western Blot, Recombinant, Purification, Positive Control

    Low-temperature growth of E. coli transformants harboring the icdII gene. (Upper panel) E. coli XL1-Blue was transformed with pMSF8 or pMS42. (Lower panel) E. coli DEK2004 was used as the host and transformed with the indicated plasmid. pBluescript was used as a vector. E. coli transformants harboring each plasmid were streaked on MOPS-succinate or MOPS-succinate-yeast extract agar plates and incubated at 10 or 15°C. Photographs were taken 2 weeks after the inoculation.

    Journal: Journal of Bacteriology

    Article Title: cis-Acting Elements Responsible for Low-Temperature-Inducible Expression of the Gene Coding for the Thermolabile Isocitrate Dehydrogenase Isozyme of a Psychrophilic Bacterium, Vibrio sp. Strain ABE-1

    doi:

    Figure Lengend Snippet: Low-temperature growth of E. coli transformants harboring the icdII gene. (Upper panel) E. coli XL1-Blue was transformed with pMSF8 or pMS42. (Lower panel) E. coli DEK2004 was used as the host and transformed with the indicated plasmid. pBluescript was used as a vector. E. coli transformants harboring each plasmid were streaked on MOPS-succinate or MOPS-succinate-yeast extract agar plates and incubated at 10 or 15°C. Photographs were taken 2 weeks after the inoculation.

    Article Snippet: E. coli DEK2004, a mutant defective in icd (a gift from Peter Thorsness), or E. coli XL1-Blue (Stratagene) was used as a host for transformation by icd genes.

    Techniques: Transformation Assay, Plasmid Preparation, Incubation

    Growth of E. coli transformants harboring 5′-deleted icdII or an icdII-icdI fusion gene. (A) E. coli DEK2004 was transformed with vector [pBluescript KS(+)] (○), pMS42 (▿), pIS202 (▴), or pMSF8 (▵). (B) E. coli DEK2004 was transformed with pTS601 (⧫), pTS602 (◊), or pSS512 (□). As a control, E. coli XL1-Blue, which has its own icd gene, was also transformed with the vector (●). Other symbols are the same as in panel A. Each transformant, precultured in LB medium at 37°C for 16 h, was inoculated in fresh LB medium and cultured at 15 or 37°C. (C) Specific activity of IDH determined with crude extract prepared from each transformant. Details of assay conditions are described in Materials and Methods. Note the thermolability of IDH-II at 37°C.

    Journal: Journal of Bacteriology

    Article Title: cis-Acting Elements Responsible for Low-Temperature-Inducible Expression of the Gene Coding for the Thermolabile Isocitrate Dehydrogenase Isozyme of a Psychrophilic Bacterium, Vibrio sp. Strain ABE-1

    doi:

    Figure Lengend Snippet: Growth of E. coli transformants harboring 5′-deleted icdII or an icdII-icdI fusion gene. (A) E. coli DEK2004 was transformed with vector [pBluescript KS(+)] (○), pMS42 (▿), pIS202 (▴), or pMSF8 (▵). (B) E. coli DEK2004 was transformed with pTS601 (⧫), pTS602 (◊), or pSS512 (□). As a control, E. coli XL1-Blue, which has its own icd gene, was also transformed with the vector (●). Other symbols are the same as in panel A. Each transformant, precultured in LB medium at 37°C for 16 h, was inoculated in fresh LB medium and cultured at 15 or 37°C. (C) Specific activity of IDH determined with crude extract prepared from each transformant. Details of assay conditions are described in Materials and Methods. Note the thermolability of IDH-II at 37°C.

    Article Snippet: E. coli DEK2004, a mutant defective in icd (a gift from Peter Thorsness), or E. coli XL1-Blue (Stratagene) was used as a host for transformation by icd genes.

    Techniques: Transformation Assay, Plasmid Preparation, Cell Culture, Activity Assay

    Optimization of expression conditions and screening background. (A) Colonies of E . coli BL21 transformed with TN-XXL cloned into the vector pRSETB (exposure time 4s, gain 2x, binning 1). Scale bar, 10 mm (B) Colonies of E . coli XL1 transformed with TN-XXL cloned into pRSETB and imaged under the same conditions. (C) Mean fluorescence intensities ± SD of colonies transformed with TN-XXL in either BL1 or XL1, imaged under identical conditions (n BL21 = 312, n XL1 = 558). (D) ΔR/R ± SD of E . coli colonies expressing TN-XXL cloned into BL21 or XL1 (n BL21 = 19, n XL1 = 15). (E) Auto-fluorescence of agar plate versus white blotting paper imaged under identical conditions with filters used for FRET imaging. Error bars indicate SD. (F) Basal ratio valuesR 0 ± SD of XL1 colonies transformed with the FRET sensor TN-XXL and imaged on agar plate or on filter paper. (n plate = 149 colonies, n blotting paper = 90 colonies) (G) ΔR/R ± SD of the same colonies following calcium application.

    Journal: PLoS ONE

    Article Title: Large Scale Bacterial Colony Screening of Diversified FRET Biosensors

    doi: 10.1371/journal.pone.0119860

    Figure Lengend Snippet: Optimization of expression conditions and screening background. (A) Colonies of E . coli BL21 transformed with TN-XXL cloned into the vector pRSETB (exposure time 4s, gain 2x, binning 1). Scale bar, 10 mm (B) Colonies of E . coli XL1 transformed with TN-XXL cloned into pRSETB and imaged under the same conditions. (C) Mean fluorescence intensities ± SD of colonies transformed with TN-XXL in either BL1 or XL1, imaged under identical conditions (n BL21 = 312, n XL1 = 558). (D) ΔR/R ± SD of E . coli colonies expressing TN-XXL cloned into BL21 or XL1 (n BL21 = 19, n XL1 = 15). (E) Auto-fluorescence of agar plate versus white blotting paper imaged under identical conditions with filters used for FRET imaging. Error bars indicate SD. (F) Basal ratio valuesR 0 ± SD of XL1 colonies transformed with the FRET sensor TN-XXL and imaged on agar plate or on filter paper. (n plate = 149 colonies, n blotting paper = 90 colonies) (G) ΔR/R ± SD of the same colonies following calcium application.

    Article Snippet: Bacterial plate screening Libraries were transformed into E .coli XL1-Blue cells or E .coli BL21-Gold cells (both Stratagene) and plated on LB agar plates containing ampicillin (100 μg/ml) with a desired colony density of ~700–800 colonies per plate.

    Techniques: Expressing, Transformation Assay, Clone Assay, Plasmid Preparation, Fluorescence, Imaging

    Permeabilizing E . coli to exogenous calcium (A) Average ΔR/R ± SD of a FRET sensor (Twitch-1) expressed in XL1 cells pretreated with poly-L-lysine and ionomycin and subjected to different concentrations of extracellular solution calcium (n = 6 colonies per concentration). (B-C) FRET trace (± standard error of the mean, grey) and individual donor and acceptor traces (± standard error of the mean, grey) of colonies of E . coli XL1 transformed with TN-XXL. Arrows indicate when calcium was applied. (n 100mM = 197 colonies, n 1M = 222 colonies). (D-E) Representative ΔR/R values following calcium application (100 mM) of colonies pretreated with different concentrations of either poly-L-lysine (PL) or ionomycin (IM), while the concentration of the second substance was kept constant. Grey dashed lines represent signal changes after the application of poly-L-lysine or ionomycin, black lines represent additional signal change after the application of calcium, and dark lines indicate total signal changes.

    Journal: PLoS ONE

    Article Title: Large Scale Bacterial Colony Screening of Diversified FRET Biosensors

    doi: 10.1371/journal.pone.0119860

    Figure Lengend Snippet: Permeabilizing E . coli to exogenous calcium (A) Average ΔR/R ± SD of a FRET sensor (Twitch-1) expressed in XL1 cells pretreated with poly-L-lysine and ionomycin and subjected to different concentrations of extracellular solution calcium (n = 6 colonies per concentration). (B-C) FRET trace (± standard error of the mean, grey) and individual donor and acceptor traces (± standard error of the mean, grey) of colonies of E . coli XL1 transformed with TN-XXL. Arrows indicate when calcium was applied. (n 100mM = 197 colonies, n 1M = 222 colonies). (D-E) Representative ΔR/R values following calcium application (100 mM) of colonies pretreated with different concentrations of either poly-L-lysine (PL) or ionomycin (IM), while the concentration of the second substance was kept constant. Grey dashed lines represent signal changes after the application of poly-L-lysine or ionomycin, black lines represent additional signal change after the application of calcium, and dark lines indicate total signal changes.

    Article Snippet: Bacterial plate screening Libraries were transformed into E .coli XL1-Blue cells or E .coli BL21-Gold cells (both Stratagene) and plated on LB agar plates containing ampicillin (100 μg/ml) with a desired colony density of ~700–800 colonies per plate.

    Techniques: Concentration Assay, Transformation Assay

    Isotype-specific phage enrichment during subsequent rounds of affinity selection. The number of phage was determined as colony-forming units (CFU) after infection of freshly grown Escherichia coli XL1 Blue cells. Notably, the absolute number of eluted phage significantly depends on the isotype library used for screening.

    Journal: Immunology

    Article Title: Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin

    doi: 10.1046/j.1365-2567.2003.01728.x

    Figure Lengend Snippet: Isotype-specific phage enrichment during subsequent rounds of affinity selection. The number of phage was determined as colony-forming units (CFU) after infection of freshly grown Escherichia coli XL1 Blue cells. Notably, the absolute number of eluted phage significantly depends on the isotype library used for screening.

    Article Snippet: Bound phage were eluted with 0·5 ml of 0·1- m HCl (pH 2·2 adjusted with solid glycine), neutralized with 60 µl of 1- m Tris–HCl (pH 8·0) and used for infection of freshly cultured, exponentially growing E. coli XL1 Blue cells.

    Techniques: Selection, Infection